Human polymorphonuclear leukocyte elastase (PMNE) cleaves human fibrinogen at multiple sites. Cleavage of the A.alpha. chain at A.alpha.(Val.sup.360 -Ser.sup.361) generates a stable product as indicated by its presence in biological fluids. A radioimmunoassay (RIA) based on the A.alpha.(Val.sup.360) epitope of this cleavage site has been developed which allows the evaluation of the potency of elastase inhibitors to inhibit formation of cleavage products containing this neoepitope in a variety of in vitro cell biological situations. The RIA detects an endogenous A.alpha.(Val.sup.360) in normal human plasma and at elevated concentrations in cystic fibrosis plasma and in rheumatoid arthritis synovial fluid samples.
Human leukocyte proteinases, elastase in particular, have been implicated in the pathogenesis of various connective tissue destructive diseases including pulmonary emphysema, chronic bronchitis, cystic fibrosis, bronchiectasis, acute respiratory distress syndrome, arthritis, glomerulonephritis, psoriasis, vasculitis and atherosclerosis. Elastase may also be involved in other lung diseases such as PiZZ emphysema and infantile respiratory distress syndrome and in myelogenous leukemia and gout. Elastase, a serine proteinase active at neutral pH, is stored in the azurophilic granules of polymorphonuclear leukocytes and in lysosomes of transient mononuclear leukocytes. Elastase as used herein is defined as elastase derived from leukocytes. Leukocyte as used herein includes neutrophils, macrophages and monocytes. Cellular elastase is released in tissues when the cell encounters matter (such as a pathogen) to be phagocytized or undergoes cellular autolysis. Tissue pathology occurs when the released elastase encounters endogenous connective tissue substrates such as elastin, proteoglycan, types III and IV collagen and fibrinonectin. In addition to connective tissue components, human leukocyte elastase hydrolyzes various plasma proteins including immunoglobulins, fibrin, fibrinogen and complement proteins.
Extracellular PMNE activity is controlled by circulating or locally produced elastase inhibitors. Circulating elastase inhibitors include alpha-1-proteinase inhibitor and alpha-2-macroglobulin. Alpha-1-proteinase inhibitor is the most prevalent regulator of endogenous elastase activity and blocks enzyme activity by complexing with the elastase so that it is unable to act on other substrates. The enzyme-inhibitor complexes are then cleared from the tissues and circulation.
Elastase and the other leukocyte associated proteinases have also been implicated in the alternate fibrinolytic pathway responsible for the dissolution of fibrinogen and fibrin. The fibrinolytic activity may be physiologically important in the thrombi reduction but may also lead to pathological conditions related to blood coagulation.
A thorough understanding of the role of leukocyte elastase in both physiological and pathological states has been hampered by the absence of a quick, efficient and reliable assay for detecting enzymatic activity in vivo with a natural substrate such as human fibrinogen. Direct measurement of the enzymatic activity of released PMNE is difficult because the released enzyme rapidly binds to the substrate or to serum proteinase inhibitors. Bound enzyme may be quickly removed from the circulation. Immunoassay determinations of released enzyme do not distinguish between free enzyme and enzyme bound to inhibitor, and determinations of enzyme-inhibitor complexes have the disadvantage that they detect only the inactivated enzyme.
Since direct detection of enzymatically active PMNE in physiological fluids has not proven reliable, alternate assay procedures dependent upon the detection of PMNE cleavage products have been designed. Two of these methods have used elastase-cleaved products of elastin as markers. One method measured urinary desmosine, a major cross-linking amino acid of elastin, by radioimmunoassay. No consistent difference has been observed between healthy individuals and chronic lung disease patients. A similar result has been reported using ELISA measurements of elastase-generated elastin-derived peptides in serum of lung disease patients and normal controls.
Assays designed to detect plasmin-generated fibrinogen degradation products (FDP) have been used to detect fibrinogen cleavage products. Early assays utilized antibodies reactive with human FDP prepared by human plasmin digestion of human fibrinogen at room temperature for 12 to 36 hours, U.S. Pat. No. 3,912,805. The antibodies are attached to red blood cells and are used in passive agglutination assays to detect FDP. Antibodies to purified FDP fragments such as fragments D and E have also been employed to identify and quantify fibrinogen degradation products, U.S. Pat. No. 4,090,846. The assay consisted of an indirect method in which human serum or urine were mixed with the antiserum to the purified D and E fragments, to which was added FDP bound to latex beads. Other fibrinogen fragments have been used to produce antibody that can be used to detect the fragments in blood. Plasminogen derived fragments such as Fg-E have been described by Chen and Schulof, Thrombos. Res. 16: 601-615 (1979) and antibodies to the fragment have been used in radioimmunoassay to detect the fragment in plasma.
Fibrinogen degradation products prepared by digesting human fibrinogen with PMNE have been used to develop assays for evaluating elastase cleavage products in fluids. Human fibrinogen was digested with PMNE at 1:50 (w/w) enzyme-to-substrate ratio for 24 hrs. at 37.degree. C. The predominant fragment was a D-like fragment of 80,000 MW and designated D.sub.e. Antibody to this fragment retained its capacity to recognize the D.sub.e in radioimmunoassays after adsorption with intact fibrinogen and plasmin degradation products. Plow et al. suggested that this indicated the recognition of an elastase-elicited neoantigen that was only expressed by elastase degradation products. When elastase degradation products were added to normal plasma, the elastase-elicited neoantigen could be quantitatively detected in radioimmunoassay. The principal disadvantage of the fibrinogen degradation product assay procedures is that the material being assayed is defined only operationally. The sites of PMNE (or plasmin) cleavage have not been adequately defined. Consequently, the specificity of the antisera cannot be rigorously investigated and the exact nature of the products measured in the immunoassay is unknown.
Immunoassays designed to detect products of thrombin cleavage of fibrinogen have been described in several animal species. For example, a radioimmunoassay has been described which identifies the peptide F-CB1.alpha. from the amino end of bovine fibrinogen A.alpha. chain. Thrombin cleavage of F-CB1 yielded two fragments: fibrinopeptide A (residues A.alpha.1-16; residues 1-19; equivalent to human residues 1-16) and the carboxy-terminal fragment Th2 (residues 20-54; equal to human residues 17-51).
Fibrinopeptide A (FPA), the 1-16 amino acid residue of the A.alpha. chain (A.alpha. 1-16), is released by thrombin cleavage and have been used to produce antibodies which can be employed to assay for the fibrinogen cleavage product FPA. Antibodies reactive with FPA were used to study the human elastase cleavage products of human fibrinogen. It was found that the elastase degradation products were different from the thrombin degradation products as determined by radioimmunoassays specific for FPA. The elastase cleavage product was slightly larger than FPA and could be distinguished from FPA by immunological means.
Antibody specifically reactive with FPA has been used to evaluate in vivo human leukocyte elastase activity. Weitz et al. found that leukocyte elastase cleaves fibrinogen into a fibrinopeptide A-containing fragment which can be used as an index of elastase activity. Human leukocyte elastase cleaves a 21 amino acid peptide, fibrinogen A.alpha. 1-21 from the amino terminus of the alpha chain of human fibrinogen. This A.alpha. 1-21 peptide contains the entire FPA peptide. The Weitz assay does not directly measure the elastase cleavage product, but measures FPA, A.alpha. 1-16, which is the normal human thrombin cleavage product of human fibrinogen. The Weitz two step radioimmunoassay utilized fresh human plasma, ethanol treated, presumably totally fibrinogen depleted, either treated or untreated with human thrombin. The difference in immunoreactive FPA, A.alpha. 1-6, between the thrombin treated and untreated samples is attributed to the amount of A.alpha. 1-21 present in the original sample. This assay does not directly measure an elastase cleavage product in body fluids and requires a two-step process to detect what appears to be a single elastase cleavage product.
The development of assays to determine the biochemical efficacy and potency of elastase inhibitors has evolved around the need to measure the activity of PMNE under a variety of circumstances in man. The evaluation of the extracellular activity of PMNE in both physiological and pathological states has been hampered by the lack of a specific assay to detect its activity in vivo. The direct measurement of the enzymatic activity of extracellular PMNE is difficult because the released enzyme binds to substrates and to plasma proteinase inhibitors almost immediately upon its release from PMN. Detection of enzyme by immunoassays does not distinguish between free enzyme and enzyme bound to inhibitors; the disadvantage of immunoassays that measure levels of enzyme inhibitors is that they detect only the inactivated enzyme.
Human fibrinogen is a hetero-dimeric glycoprotein consisting of 3 non-identical chains, A.alpha., B.beta. and .gamma.. PMNE cleaves human fibrinogen at multiple sites. Primary sites of cleavage include A.alpha.(Val.sup.21 -Glu.sup.22), A.alpha.(Val.sup.360 -Ser.sup.361), A.alpha.(Val.sup.450 -Ile.sup.451), A.alpha.(Val.sup.464 -Thr.sup.465), A.alpha.(Met.sup.476 -Asp.sup.477), A.alpha.(Thr.sup.568 -Ser.sup.569), .gamma.(Thr.sup.305 -Ser.sup.306), .gamma.(Val.sup.347 -Tyr.sup.348) and .gamma.(Ala.sup.357 -Ser.sup.358). We have developed two antipeptide antibodies, one of which specifically measures PMNE hydrolysis of fibrinogen at the A.alpha.(Val.sup.21 -Glu.sup.22) position to release a 21 residue N-terminal peptide, and a second which measures cleavage at A.alpha.(Val.sup.360 -Ser.sup.361), to release a 250 residue C-terminal fragment (FIG. 1). The A.alpha.(Val.sup.360) carboxyl terminal fragment remains associated with the .beta. and .gamma. chains of fibrinogen due to the disulfide network of the protein. Neither of the 2 specific antisera recognize intact fibrinogen. Both of these RIA allow the evaluation of the potency of PMNE inhibitors, such as elastase inhibitors, to inhibit fibrinopeptide neoepitope generation in whole blood stimulated with the calcium ionophore A23187. However, a major disadvantage of the A.alpha.(Val.sup.21) assay is the rapid in vivo clearance and metabolism of the peptide neoepitope A.alpha.(Val.sup.21) (t.sub.1/2 of 30 sec in both the dog and rhesus monkey). In an extensive series of experiments we were unable to detect the A.alpha.(Val.sup.21) neoepitope in normal human plasma or in plasma samples from PiZZ individuals, nor in plasma from from patients with cystic fibrosis, emphysema or chronic bronchitis.
The A.alpha.(Val.sup.360) neoepitope is associated with a large protein fragment which results in a substantially slower clearance rate than A.alpha.(Val.sup.21) peptide. Utilizing the A.alpha.(Val.sup.360) RIA an endogenous signal is detected in normal human plasma. This signal is elevated in cystic fibrosis plasma samples. In addition, levels of A.alpha.(Val.sup.360) neoepitope are elevated in both rheumatoid arthritis and gout synovial fluid but not in osteoarthritis synovial fluid.
This assay may be readily incorporated into a diagnostic kit for measuring PMNE activity in man. The assay measures the potency of elastase inhibitors in plasma, sputum and synovial fluid following oral administration of drug. In a Phase 1 clinical study with elastase inhibitors, the A.alpha.(Val.sup.360) RIA in combination with assays of AAPVase activity and L-740,447 formation in blood, would provide data indicating the extent of inhibition of enzyme activity within cells (AAPVase in isolated PMN), generation of PMNE:elastase inhibitor complexes (L-740,447 assay) in whole blood and upon its release from cells to act on extracellular substrate [A.alpha.(Val.sup.360) neoepitope]. In subsequent clinical studies in cystic fibrosis patients, lowering of elevated levels of A.alpha.(Val.sup.360) would provide direct evidence of inhibition of PMNE activity.
The present invention is a radioimmunoassay (RIA) based on the A.alpha.(Val.sup.360) epitope which allows the evaluation of the potency of elastase inhibitors to inhibit formation of cleavage products containing this neoepitope in a variety of in vitro cell biological situations. This new RIA detects an endogenous A.alpha.(Val.sup.360) signal in normal human plasma and at elevated levels in cystic fibrosis plasma and in rheumatoid arthritis synovial fluid samples. The assay procedure is a single step assay which allows for the rapid and reproducible detection of a PMNE-specific cleavage peptide.